Bacillus licheniformis JSF-9 and its composite bacterial agent and application

Abstract

The present invention relates to the field of microbial technology, and in particular to Bacillus licheniformis JSF-9, a composite bacterial agent and applications thereof. The strain JSF-9 of the present invention is isolated by a research team from the rectal contents of healthy cattle. Testing shows that the strain has good β-1,4-glucanase production ability and has antibacterial effects on Staphylococcus aureus, Salmonella typhimurium and Shigella flexneri. The strain has good fermentation ability for sugarcane bagasse. For example, after the strain is prepared into a mixed bacterial agent with Bacillus licheniformis MLF-1 and Bacillus sabdariffa MLL-5, the fermentation ability of the mixed bacterial agent for sugarcane bagasse is better. It has been verified that the composite bacterial agent can effectively inhibit the reproduction of harmful bacteria and thus prolong the shelf life of fermented feed. Animal feeding experiments have found that the nutritional value and disease resistance of sugarcane bagasse fermented with the composite bacterial agent are significantly improved. Adding 10%-15% to the basal diet has the effect of reducing costs and increasing efficiency in feeding Jersey cattle.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Bacillus licheniformis JSF-9 and its compound bacterial agents and applications. Background Technology

[0002] Sugarcane is the main raw material for white sugar production in southern my country and one of the country's major sugar crops. How to effectively utilize sugarcane bagasse, a waste product of white sugar production, is a key research focus in our region. Currently, sugarcane bagasse is mainly used for silage, organic fertilizer, and even beverage production. Many microorganisms are used in silage production, with lactic acid bacteria being the most common. Lactic acid bacteria are not only fermenting bacteria but also probiotics that can effectively regulate animal intestines. Existing technologies have also reported the use of Bacillus subtilis for sugarcane bagasse fermentation. However, some Bacillus subtilis can alter the intestinal flora environment when applied to animals, leading to diarrhea. Therefore, when screening Bacillus subtilis, we need to pay more attention to its antibacterial properties against intestinal pathogens to effectively inhibit their growth. In addition, Bacillus subtilis has a strong ability to decompose cellulose, providing a new technical route for the effective utilization of sugarcane bagasse. Therefore, it is necessary to screen and verify its cellulase decomposition ability to select Bacillus subtilis suitable for sugarcane bagasse fermentation, thereby improving the utilization rate of sugarcane bagasse by microorganisms.

[0003] Therefore, we need to conduct extensive screening and validation of microorganisms, develop probiotics suitable for fermentation of sugarcane bagasse substrate, produce sugarcane bagasse fermented feed, and verify the feasibility of feeding this feed to animals. Summary of the Invention

[0004] In view of the above, it is necessary to verify the antibacterial properties and cellulase degradation ability, screen suitable Bacillus species, develop microorganisms that can effectively ferment sugarcane bagasse substrate, provide theoretical support for the production of sugarcane bagasse fermented feed, and preliminarily verify the feasibility of sugarcane bagasse fermented feed in animal feeding.

[0005] To achieve the above objectives, this invention has screened out a new strain: Bacillus licheniformis (Bacillus licheniformis) Bacillus paralicheniform JSF-9, its classification name is: Bacillus paralicheniformis JSF-9, Chinese classification name: Bacillus licheniformis JSF-9, accession number GDMCC NO: 65786; this strain is deposited at Guangdong Provincial Center for Microbial Culture Collection, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 14, 2025.

[0006] The present invention also includes the Bacillus licheniformis ( Bacillus paralicheniformis JSF-9 compound microbial agent.

[0007] Furthermore, the compound microbial agent also includes Bacillus hygroscopicus (B. hygroscopicus). Bacillus altitudine s) MLF-1 and / or Bacillus safranin ( Bacillus safensis MLL-5; the Highland Bacillus ( Bacillus height s) MLF-1, its classification name is: Altitude Bacillus MLF-1, classified in Chinese as *Bacillus salina* MLF-1, with accession number GDMCC NO: 65789, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 14, 2025. The *Bacillus salina* strain described is... Bacillus safensis MLL-5, its classification name is: Bacillus safensis MLL-5, classified in Chinese as *Bacillus sabolicus* MLL-5, with accession number GDMCC NO: 65790, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on February 10, 2025.

[0008] Furthermore, the compound bacterial agent is composed of Bacillus hygroscopicus (B. hygroscopicus). Bacillus altitudine s)MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis JSF-9 and Bacillus safranin ( Bacillus safensis MLL-5 was prepared by mixing in a volume ratio of (1-2):(2-6):(1-3).

[0009] Furthermore, the compound bacterial agent is composed of Bacillus hygroscopicus (B. hygroscopicus). Bacillus altitudine s)MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis JSF-9 and Bacillus safranin ( Bacillus safensis MLL-5 was prepared by mixing in a volume ratio of 1:6:3.

[0010] The present invention also includes the Bacillus licheniformis ( Bacillus paralicheniformis Application of JSF-9 or the aforementioned compound microbial agent in the preparation of sugarcane bagasse fermented feed.

[0011] The present invention also includes the Bacillus licheniformis ( Bacillus paralicheniformis Application of JSF-9 in the production of β-1,4-glucanase.

[0012] The present invention also includes the Bacillus licheniformis ( Bacillus paralicheniformis The application of JSF-9 in the preparation of an antibacterial agent for intestinal pathogens, wherein the intestinal pathogen is Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus Salmonella typhimurium ( Salmonella typhimurium.) and / or Shigella flexneri ( Shigella flexible ).

[0013] The present invention also includes the Bacillus licheniformis ( Bacillus paralicheniformis Fermented feed containing JSF-9 or the aforementioned compound microbial agent, wherein the fermented feed is prepared by: Bacillus licheniformis (… Bacillus paralicheniform The product is obtained by inoculating sugarcane bagasse with JSF-9 or compound microbial agent at an inoculation rate of 100 ml / kg and fermenting for 30 days.

[0014] The present invention also includes the application of the fermented feed in feeding Jersey cattle.

[0015] The present invention has the following beneficial effects: 1. The strain JSF-9 of the present invention was isolated from healthy rectal contents by the research group. After testing, the strain has a good ability to produce β-1,4-glucanase and has antibacterial effects against Staphylococcus aureus, Salmonella typhimurium and / or Shigella flexneri. The strain has a good fermentation ability for sugarcane bagasse. When the strain is prepared into a mixed microbial agent with Bacillus hygroscopicus MLF-1 and Bacillus salsa MLL-5, the fermentation ability of sugarcane bagasse is even better. And after intestinal flora verification, it was found that the compound microbial agent can effectively inhibit the reproduction of harmful bacteria, thereby extending the shelf life of fermented feed. After feeding Jersey cattle with fermented feed prepared by the compound microbial agent, it was found that the fermented sugarcane bagasse of the compound microbial agent was significantly improved in terms of both nutritional value and disease resistance. Adding 10%-15% to the basic diet can reduce costs and increase efficiency in feeding Jersey cattle.

[0016] 2. This application also conducted orthogonal experiments to determine the optimal volume ratio of the compound microbial agent. The use of this compound microbial agent in the synergistic fermentation of sugarcane bagasse further improved the utilization rate of feed. The research on this strain, compound microbial agent and fermentation method provides good support and technical means for solving the waste of sugarcane processing enterprises. Attached Figure Description

[0017] Figure 1 This is a colony morphology diagram of strain MLF-1.

[0018] Figure 2 This is a colony morphology diagram of strain JSF-9.

[0019] Figure 3 This is a colony morphology diagram of strain MLL-5.

[0020] Figure 4 The results show the destaining of strain JSF-9 on Congo red-stained sodium carboxymethyl cellulose selection medium.

[0021] Information on the preservation of biological materials

[0022] The strain information preserved in this application is: Bacillus hygroscopicus ( Bacillus altitudine s) MLF-1, its classification name is: Altitude Bacillus MLF-1, classified in Chinese as Bacillus hygroscopicus MLF-1, with accession number GDMCC NO: 65789, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 14, 2025.

[0023] The strain information preserved in this application is: Bacillus licheniformis ( Bacillus paralicheniformis JSF-9, its classification name is: Bacillus paralicheniformis JSF-9, Chinese classification name: Bacillus licheniformis JSF-9, accession number GDMCC NO: 65786; this strain is deposited at Guangdong Provincial Center for Microbial Culture Collection, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 14, 2025.

[0024] The strain information preserved in this application is: Bacillus safranin ( Bacillus safensis MLL-5, its classification name is: Bacillus safensis MLL-5, classified in Chinese as *Bacillus sabolicus* MLL-5, with accession number GDMCCNO: 65790, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on February 10, 2025. Detailed Implementation

[0025] All features disclosed in this specification, or steps in all methods or processes disclosed herein, may be combined in any way, except for mutually exclusive features and / or steps.

[0026] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0027] Example 1

[0028] This example demonstrates the isolation and identification of the bacterial strain.

[0029] Isolation and purification of the strain: A total of 67 samples of rumen and rectal contents from healthy cattle were collected, placed in sterile cryovials containing glycerol, sealed with sealing film, and transported back to the laboratory in liquid nitrogen for storage at -80°C for later use. The rumen contents stored at -80°C were thawed at low temperature and serially diluted 10-fold in sterile PBS in a sterile operating table, and thoroughly shaken (vortexed) to form a 1:10 homogenate. Using sterile pipettes or micropipette tips, 10-fold serial dilutions of the samples were prepared following the same procedure, using a different sterile pipette or tip for each dilution. 100 μL of each of 3-4 appropriate dilutions was spread onto the surface of NA agar medium. After spreading, the plates were allowed to stand to allow the inoculum to be completely absorbed by the medium. The plates were then inverted and incubated at 39°C. After colonies grew, colonies of different morphologies were picked and purified using the streak plating method (2-3 times). The purified strains were preserved in magnetic bead culture tubes, resulting in a total of 256 purified strains. The purified strains were inoculated onto sodium carboxymethyl cellulose (CCC) agar plates and incubated at 39°C for 18-24 h. After staining with 0.1% Congo red solution for 15 min, the stain was discarded, and then 1 mol / L NaCl solution was added for destaining for 30 min. Cellulase-producing strains were determined based on the ratio of the clear zone diameter (D) to the colony diameter (d) (D / d), with each strain being replicated three times. A total of 15 cellulase-producing strains were screened. Strains with a high ratio of clear zone size to colony size were selected, and the enzyme production conditions were optimized. The specific results are shown in Table 1.

[0030]

[0031] Three strains with large D / d ratios were selected from Table 1 for strain identification.

[0032] (1) Identification of strain MLF-1: ① Morphological identification, such as Figure 1 As shown: MLF-1 strain was inoculated onto LB agar plates and cultured for 24 hours. Colonies were round, white, 1.5–3.5 mm in diameter, with irregular edges, thick and moist, and easy to pick. ② DNA was extracted from the strain and sequenced. Molecular identification was performed, and the strain's DNA sequence was amplified. PCR amplification and sequencing of the 16S region were conducted using primers: forward sequence 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse sequence 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'. After sequencing the PCR product, the obtained 16S sequence is shown in SEQ ID NO: 1 of the sequence listing. BLAST alignment and housekeeping gene testing confirmed that this strain is related to… Altitude Bacillus Based on the close phylogenetic relationship and morphological identification, this strain was classified and named... Altitude Bacillus .

[0033] (2) Identification of strain JSF-9: ① Morphological identification, such as Figure 2 As shown: JSF-9 strain was inoculated onto LB agar plates and cultured for 24 hours. Colonies were round, milky white, smooth, and had regular edges. ② After extracting and sequencing the strain's DNA, molecular identification was performed. The DNA sequence of the strain was amplified, and the 16S region was amplified and sequenced by PCR using primers: forward sequence 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse sequence 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'. After sequencing the PCR product, the obtained 16S sequence is shown in SEQ ID NO: 2 of the sequence listing. BLAST alignment and housekeeping gene testing confirmed that this strain is related to... Bacillus paralicheniformis Based on the close phylogenetic relationship and morphological identification, this strain was classified and named... Bacillus paralicheniformis .

[0034] (3) Identification of strain MLL-5: ① Morphological identification, such as Figure 3 As shown: MLL-5 strain was inoculated onto LB agar plates and cultured for 24 hours. Colonies were round, yellowish-white, smooth, and had regular edges. ② After extracting and sequencing the strain's DNA, molecular identification was performed. The DNA sequence of the strain was amplified, and the 16S region was amplified and sequenced by PCR using primers: forward sequence 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse sequence 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'. After sequencing the PCR product, the obtained 16S sequence is shown in SEQ ID NO: 3 of the sequence listing. BLAST alignment and housekeeping gene testing confirmed that this strain is related to... Bacillus safensis Based on the close phylogenetic relationship and morphological identification, this strain was classified and named... Bacillus safensis .

[0035] Example 2

[0036] This example describes the enzyme production of strain JSF-9 and the determination of optimal enzyme production conditions.

[0037] (1) Seed culture medium: 10.0g peptone, 5.0g yeast extract, 10.0g sodium chloride, 1L distilled water, natural pH, sterilized at 121℃ for 20min.

[0038] (2) Liquid fermentation medium: 50g sugarcane bagasse, 10.0g peptone, 10.0g sodium chloride, 1L distilled water, natural pH, sterilized at 121℃ for 20min.

[0039] (3) Inoculate strain JSF-9 into seed liquid culture medium and culture at 39℃ and 160r / min with shaking until OD. 600The concentration was 0.6, and the seed solution was obtained.

[0040] (4) The seed culture was inoculated into 250ml Erlenmeyer flasks containing 100ml of liquid fermentation medium at inoculation rates of 3%, 5%, 7%, and 9% (V / V). Sugarcane bagasse was used as an inducer to induce the strain to secrete cellulase. The fermentation was carried out at 39℃ for 2, 3, 4, and 5 days at a shaking speed of 160r / min. The fermentation broth was centrifuged at 4℃ and 6000r / min for 10 minutes, and the supernatant was collected as the crude enzyme solution. The enzyme production of strain JSF-9 was finally obtained as follows: Figure 4 As shown in the figure: strain JSF-9 can produce a clear zone on Congo red-stained sodium carboxymethyl cellulose selection medium, indicating that the strain can produce β-1,4-glucanase. The specific enzyme production is shown in Table 2.

[0041]

[0042] As shown in Table 2, strain JSF-9, with a 9% inoculum and 3 days of shake-flask fermentation, showed the best enzyme production of endo-β-1,4-glucanase at 2546.29 U / ml; with a 9% inoculum and 4 days of shake-flask fermentation, exo-β-1,4-glucanase produced the best enzyme at 615.93 U / ml.

[0043] Example 3

[0044] This example demonstrates the antibacterial effect of strain JSF-9 against common enteropathogenic bacteria.

[0045] The effectiveness of strain JSF-9 against Staphylococcus aureus was determined using the perforated agar diffusion method. Staphylococcus golden ), Escherichia coli ( Escherichia coli Salmonella typhimurium ( Salmonella typhimurium. Yersinia enterocolitica ( ) Yersinia enterocolitica ), Shigella flexneri ( Shigella flexneri ), Enterobacter aerogenes ( Enterobacter aerogenes The antibacterial activity of the sample was measured in triplicate for each sample; the results are shown in Table 3.

[0046]

[0047] Note: “—” in the table indicates no inhibitory effect; different letters in the subheadings of data in the same column indicate significant differences (P<0.05); the same letter in the subheadings or no letter indicates no significant difference (P>0.05).

[0048] Table 3 shows that strain JSF-9 is effective against Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus Salmonella typhimurium ( ) , Salmonella typhimurium Salmonella typhimurium.) and / or Shigella flexneri ( Shigella flexneri It has antibacterial effects against Escherichia coli (Escherichia coli). Escherichia coli Yersinia enterocolitica ( ) Yersinia enterocolitica ) and Enterobacter aerogenes ( Enterobacter aerogenes No antibacterial effect; Staphylococcus aureus (Staphylococcus aureus) is present in the inhibition zone. Staphylococcus aureus Salmonella Typhimurium ( Salmonella typhimurium. Shigella flexneri Shigella flexneri Escherichia coli ( Escherichia coli This indicates that strain JSF-9 is effective against Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus The antibacterial effect of ) is the best, followed by Salmonella typhimurium ( Salmonella typhimurium. Then there's Shigella flexneri. Shigella flexneri ) .

[0049] Example 4

[0050] This example demonstrates the fermentation effects of different selected microbial agents on sugarcane bagasse fermented feed.

[0051] 1. Experimental Design: This experiment adopted a completely randomized experimental design, setting up 5 groups, with 3 replicates in each group: a control group (CK group) without any bacterial agent, and a group supplemented with Bacillus salsa MLL-5 (T1, Bacillus salsa 4.6×10⁻⁶). 7 cfu / ml), with the addition of Bacillus licheniformis JSF-9 group (T2, 1×10⁻⁶ cfu / ml), 7 CFU / ml Bacillus licheniformis), with the addition of Bacillus hygroscopicus MLF-1 group (T3, Bacillus hygroscopicus 6.2×10⁻⁶). 7 The sugarcane bagasse was adjusted to a moisture content of about 60% with distilled water. According to the experimental design, 100 ml / kg of the corresponding microbial agent was added, mixed evenly, and packed into polyethylene bags (230 mm × 400 mm) with one-way exhaust valves, 500 g per bag. Each treatment was replicated 3 times. The bags were sealed with a vacuum sealer and fermented at room temperature in the dark for 30 days.

[0052] 2. Measurement indicators and methods.

[0053] (1) Routine nutritional component detection and analysis: The sample was dried at 105℃ for 2 hours, and then dried at 65℃ to constant weight. The dry matter (DM) content was calculated (Zhang Liying. Feed Analysis and Feed Quality Testing Technology [M]. 2nd ed. Beijing: China Agricultural University Press, 2003.); it was pulverized and passed through a 40-mesh sieve for the determination of crude protein (CP), water soluble carbohydrates (WSC), neutral detergent fiber (NDF), and acid detergent fiber (ADF). CP, NDF, and ADF were determined using the method described in (van Soest P J, Sniffen CJ, Mertens D R. A net protein system for cattle: The rumen submodel for nitrogen [C] / / Owens F N. Protein Requirements for Cattle: Proceedings of an International Symposium. 1981. Stillwater: Oklahoma State University: 265.); WSC was determined using the anthrone-colorimetric method.

[0054] (2) Determination of fermentation quality of fermented sugarcane bagasse: Take 10g of fresh sample into an Erlenmeyer flask, add 90mL of distilled water, shake well, seal with sealing film, and place in a refrigerator at 4℃ for 24h. After filtering the extract through gauze, centrifuge at 8000r / min for 10min and collect the supernatant for the detection of fermentation parameters. pH was measured using a pH meter, and ammonia nitrogen (NH3-N), lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) were detected by high performance liquid chromatography. Total nitrogen = crude protein / 6.25.

[0055] The results obtained by using the above method are shown in Tables 4-6.

[0056]

[0057] Note: Different letters in the superscript of data from the same line indicate significant differences (P<0.05); the same letter in the superscript or no letter indicates no significant differences (P>0.05). The same applies below.

[0058] Table 5 shows that, regarding DM content, there was no significant difference between CK and experimental groups T1, T2, and T3, indicating that Bacillus subtilis can ferment with minimal dry matter loss and effectively preserve the nutrients in sugarcane bagasse. Regarding CP content, the CP content of group T2 was significantly higher than other experimental groups (P<0.05), while there were no significant differences between groups T1 and T3 and CK (P>0.05), indicating that fermentation by strain JSF-9 can significantly increase the protein content of sugarcane bagasse. Regarding WSC, after fermentation, the WSC content of each group was significantly higher than the original content. The content of 1.18% DM in the feed was reduced; the ADF and NDF contents of the T1, T2 and T3 experimental groups were significantly lower than those of the CK group (P<0.05), indicating that Bacillus has a good ability to degrade cellulose. In terms of degradation rate, the content of T2 group was the lowest and the difference from other experimental groups was significant (P<0.05). The above results show that strain JSF-9 has a good fermentation ability for sugarcane bagasse. After fermentation, it can effectively increase the protein content of fermented sugarcane bagasse and effectively degrade the neutral detergent fiber and acid detergent fiber content of sugarcane bagasse.

[0059]

[0060] Table 6 shows that, in terms of pH value, CK and experimental groups T1, T2, and T3 all had pH values ​​below 4.2, but the differences between groups were not significant (P>0.05). pH and organic acidity are important indicators reflecting the quality of feed fermentation. High-quality fermented feed generally has a pH below 4.2. In this experiment, the pH values ​​of the three additive treatment groups were... All pH values ​​were below 4.2, indicating that the pH of the above feeds reached good quality after fermentation. Regarding LA content, the LA content in the CK group was significantly lower than that in the T1, T2, and T3 experimental groups (P<0.05). Among the T1, T2, and T3 experimental groups, the T2 experimental group had the highest LA content, followed by the T3 group, and then the T1 group. Regarding AA content, the AA content in the CK group was significantly higher than that in the T1, T2, and T3 experimental groups (P<0.05). Among the T1, T2, and T3 experimental groups, the T2 experimental group had the lowest LA content, followed by the T3 and T1 groups. Regarding PA content, the T1 group had significantly higher PA content than the CK group, followed by the T3 and T2 groups (P<0.05). Regarding BA content... The CK group had significantly higher pH values ​​than the T1, T2, and T3 groups (P<0.05), while there were no significant differences among the T1, T2, and T3 groups (P>0.05). The fermentation results of organic acids showed that although the pH values ​​of the CK group were not significantly different from those of the T1, T2, and T3 groups, the composition of organic acids differed considerably. The lactic acid content in the experimental group with added Bacillus was significantly higher than that of the CK group. This may be because Bacillus can rapidly consume oxygen, creating an anaerobic environment in a short time, which is conducive to the reproduction of lactic acid bacteria and thus increases lactic acid production. Regarding NH3-N content, there was no significant difference between the CK and T1 and T3 experimental groups (P>0.05), while the NH3-N content of the T2 experimental group was significantly lower than that of the other experimental groups.

[0061] In summary, when Bacillus licheniformis JSF-9 is used as a single inoculum, the fermentation effect on sugarcane bagasse is the best. After fermentation, it can effectively increase the protein content of fermented sugarcane bagasse, effectively degrade the neutral detergent fiber and acid detergent fiber content of sugarcane bagasse, and increase the lactic acid content while decreasing the NH3-N content.

[0062] Example 5

[0063] This example demonstrates the fermentation effect of a compound microbial agent on sugarcane bagasse fermented feed.

[0064] Based on the experimental results of Example 4, we found that among the three Bacillus strains, Bacillus licheniformis JSF-9 had the best fermentation effect on sugarcane bagasse substrate. Bacillus fulviculitis MLL-5 and Bacillus pleuropneumoniae MLF-1 had a certain fermentation effect on sugarcane bagasse, but it was not as significant as that of strain JSF-9. However, in the pathogen inhibition experiment, we found that Bacillus fulviculitis MLL-5 and Bacillus pleuropneumoniae MLF-1 had a better inhibitory effect on intestinal pathogens than strain JSF-9. Based on this, in order to improve the antibacterial function of fermented feed and further improve the quality of sugarcane bagasse fermented feed, we considered using the three groups of Bacillus strains as a compound microbial agent to inoculate sugarcane bagasse for fermentation, and used orthogonal experiments to optimize the strain ratio of the compound microbial agent. Specifically, the method was as follows: strain MLF-1, strain JSF-9 and strain MLL-5 were mixed to prepare a compound microbial agent, and then the compound microbial agent was inoculated at an inoculation amount of 100 ml / kg to prepare sugarcane bagasse fermented feed according to the method of Example 4. The effective viable count of Bacillus licheniformis JSF-9 was 1.2 × 10⁻⁶. 8 The effective viable count of Bacillus hygroscopicus MLF-1 was 3.5 × 10⁻⁶ CFU / ml. 7 The effective viable count of Bacillus salsa MLL-5 was 7.5 × 10⁻⁶ CFU / ml. 7 cfu / ml; the optimal experimental group was selected based on the content of crude protein (CP), lactic acid (LA) and ammonia nitrogen, as shown in Tables 7 and 8.

[0065]

[0066]

[0067] As shown in Table 8, regarding crude protein, experiments 1-3 and 5 showed better improvement than group T2 (2.23%) in Table 5, with experiment 3 showing the most significant improvement. Regarding lactic acid content, experiments 1-4 showed better improvement than group T2 (0.56 mg / g) in Table 6, with experiment 2 showing the most significant improvement. Regarding ammonia nitrogen content, experiments 1-4 showed better reduction than group T2 (4.63%) in Table 5, while the reduction in other experimental groups was less significant than that in group T2, with experiment 3 showing the most significant reduction. Therefore, considering all factors... The volume ratio of strains selected in experiments 1-4 to prepare the compound microbial agent showed a better fermentation effect on sugarcane bagasse, i.e., the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 was (1-2):(2-6):(1-3). In combination, although the LA content in experiment 3 was not as high as that in experiment 2, the difference between the two was not significant. Therefore, considering other indicators, we considered experiment 3 as the optimal experimental group, i.e., the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 was 1:6:3.

[0068] In terms of range, strain MLF-1 had the greatest impact on crude protein in fermented sugarcane bagasse feed, followed by strain MLL-5, while strain JSF-9 had the least impact. Strain MLF-1 had the greatest impact on lactic acid in fermented sugarcane bagasse feed, followed by strain MLL-5, while strain JSF-9 had the least impact. Strain MLF-1 had the greatest impact on ammonia nitrogen in fermented sugarcane bagasse feed, followed by strain MLL-5, while strain JSF-9 had the least impact.

[0069] Example 6

[0070] This example is a feeding experiment on Jersey cattle with fermented sugarcane bagasse feed.

[0071] Forty 7-month-old Jersey cattle in good condition and with similar weights were randomly divided into four groups of 10 each. The control group (CK) was fed a basal diet of whole-plant corn silage; experimental group 1: 10% fermented sugarcane bagasse (fermented with sterile agent) was added to the basal diet; experimental group 2: 10% fermented sugarcane bagasse (compound microbial agent added according to the proportion in Experiment 3 of Example 5) was added to the basal diet; experimental group 3: 15% fermented sugarcane bagasse (compound microbial agent added according to the proportion in Experiment 3 of Example 5) was added to the basal diet. The pre-feeding period was 7 days, the experimental period was 28 days, and the entire experiment lasted 35 days. The initial weight and final weight of the Jersey cattle were measured, and the average daily weight gain was calculated. The daily feed intake was recorded and the average daily feed intake was calculated. The feed conversion ratio was calculated based on the average daily feed intake / average daily weight gain. The number of diarrhea episodes was observed and counted (1 episode of diarrhea was recorded as 1, and 2 episodes of diarrhea were recorded as 2). The specific results are shown in Table 9.

[0072]

[0073] Note: Different lowercase letters in the superscript of data in the same column indicate significant differences (P<0.05), while the presence of the same letter or the absence of a letter indicates no significant differences (P>0.05).

[0074] As shown in Table 9, in terms of average daily weight gain, there was no significant difference between experimental groups 2 and 3 and the control group (P>0.05), but the weight gain was significantly higher than that of experimental group 1 (P<0.05). In terms of feed conversion ratio, there was no significant difference between experimental groups 2 and 3 and the control group (P>0.05), but the weight gain was significantly lower than that of experimental group 1 (P<0.05). In terms of the frequency of diarrhea, the incidence of diarrhea in experimental group 1 was higher than that in the control group, which was higher than that in experimental groups 2 and 3. This indicates that because the compound microbial agent has an inhibitory effect on a variety of intestinal bacteria, the fermented sugarcane bagasse prepared with it also has a good anti-diarrheal ability. Sugarcane bagasse feed that was not fermented with the compound microbial agent may have bred other pathogenic bacteria, resulting in a higher frequency of diarrhea than the control group. The above conclusions indicate that sugarcane bagasse fermented with compound microbial agents can effectively improve its nutritional value and replace basic feed. It is also possible that the compound microbial agent fermentation of sugarcane bagasse enhances the disease resistance and immunity of Jersey cattle, thereby improving their feed utilization rate. From the feeding results, after fermentation with compound microbial agents, both the nutritional value and disease resistance of sugarcane bagasse are significantly improved. Adding 10%-15% to the basic diet can reduce costs and increase efficiency in feeding Jersey cattle.

[0075] In summary, the Bacillus licheniformis JSF-9, which was screened by the applicant of this invention, has a good ability to produce β-1,4-glucanase and has antibacterial effects against Staphylococcus aureus, Salmonella typhimurium, and / or Shigella flexneri. Bacillus licheniformis JSF-9 has a good fermentation ability for sugarcane bagasse. After feeding Jersey cattle with the fermented sugarcane bagasse feed prepared by the compound microbial agent, it was found that both the nutritional value and disease resistance of the fermented sugarcane bagasse were significantly improved. Adding 15% to the basic diet can reduce costs and increase efficiency in feeding Jersey cattle.

[0076] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Bacillus licheniformis ( Bacillus paralicheniformis ) Application of strain JSF-9 in the preparation of an antibacterial agent for intestinal pathogens, characterized in that: The intestinal pathogen is Staphylococcus aureus ( Staphylococcus aureus ), Salmonella typhimurium ( Salmonella typhimurium ) and Shigella flexneri ( Shigella flexneri ); the Bacillus licheniformis Bacillus paralicheniformis The deposit number of strain JSF-9 is GDMCC NO:65786.

2. A composite bacterial agent comprising the Bacillus licheniformis strain JSF-9 according to claim 1.

3. The composite bacterial agent according to claim 2, characterized in that The composite bacterial agent also includes Bacillus subtilis ( Bacillus altitudinis ) strain MLF-1 and / or Bacillus saffron ( Bacillus safensis ) strain MLL-5; the deposit number of the Bacillus subtilis strain MLF-1 is GDMCC NO: 65789; the deposit number of the Bacillus subtilis strain MLL-5 is GDMCC NO: 65790.

4. The composite bacterial agent according to claim 3, characterized in that The composite bacterial agent is prepared by mixing Bacillus subtilis strain MLF-1, Bacillus licheniformis strain JSF-9 and Bacillus saffron strain MLL-5 in a volume ratio of 1-2:2-6:1-3. The effective viable cell count of the Bacillus licheniformis strain JSF-9 is 1.2×10 8 cfu / ml, and the effective viable count of Bacillus subtilis strain MLF-1 was 3.5×10 7 cfu / ml, and the effective viable count of Bacillus serratus strain MLL-5 was 7.5×10 7 cfu / ml.

5. The composite bacterial agent according to claim 4, characterized in that The composite bacterial agent is prepared by mixing Bacillus subtilis strain MLF-1, Bacillus licheniformis strain JSF-9 and Bacillus saffron strain MLL-5 in a volume ratio of 1:6:

3.

6. Use of the Bacillus licheniformis strain JSF-9 according to claim 1 or the composite bacterial agent according to claim 2 in preparing sugarcane bagasse fermented feed.

7. Use of the Bacillus licheniformis strain JSF-9 as claimed in claim 1 in producing β-1,4-glucanase.

8. A fermented feed comprising the Bacillus licheniformis strain JSF-9 according to claim 1 or the composite bacterial agent according to claim 2, characterized in that: The preparation method of the fermented feed is as follows: inoculating the Bacillus licheniformis strain JSF-9 or the composite bacterial agent into sugarcane bagasse at an inoculation amount of 100 ml / kg and fermenting for 30 days to obtain the feed.

9. Use of the fermented feed according to claim 8 in feeding Jersey cattle.

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